This invention relates generally to biological assays, and more specifically to assay reagents labeled with fluorescent materials which reagents can be xe2x80x9ctoggledxe2x80x9d from an intramolecular dimer to a fluorescent monomer by antibody binding.
Most clinical assays (e.g., immunoassays, DNA probe assays) are heterogeneous and consist of at least two steps: the binding of an antigen to its antibody, followed by physical separation of the bound from free antigens. In some more sensitive assays (e.g., xe2x80x9cELISAxe2x80x9d or xe2x80x9cEIAxe2x80x9d) multiple steps are required. Homogeneous immunoassays, on the other hand, can distinguish between bound antigens and free ones without the need of additional separation steps. They are simple, rapid, yet more precise, more cost effective, and have the potential for total automation. For these reasons, separation-free assays are preferred in many applications such as biosensors, bioprobes and other automated instrumentation. J. P. Gosling, Clin. Chem., 36:1408-1427 (1990), D. W. Chan and M. T. Perlstein, Eds., Immunoassay, A Practical Guide (Academic Press, New York, 1987), and E. F. Ullman and P. L. Khanna, Methods in Enzymology, 74:28-60 (1981).
However, because of various technical complications homogeneous systems have been difficult to obtain, with the exception of a few assays suitable only for small molecules. J. F. Burd et al., Clin. Chem., 23:1402-1408 (1977), M. E. Jolley et al., Clin. Chem., 27:1190-1197 (1981), and D. L. Morris et al., Anal. Chem., 53:658-665 (1981).
It would be an improvement in the art to develop and characterize new fluorogenic tracer antigens that can be used as xe2x80x9creporter moleculesxe2x80x9d for the binding event without the need of separation steps and the labeling of antibodies. The development of such tracers could greatly facilitate the automation of a large array of clinical assays, especially of high molecular weight antigens. It would help reduce the operational time and cost, and make such assays more readily accessible to doctors and patients. Also, such tracers would be extremely useful for rapidly screening large numbers of recombinant antibodies generated with genetic engineering techniques, such as those described in C. F. Barbas et al., Proc. Natl. Acad. Sci. USA 89:4457-4461 (1992), R. A. Lerner et al., Science 258:1313-1314 (1992), and Marks et al. J. Biol. Chem. 267:16007-16010 (1992).
The invention includes a fluorogenic tracer antigen that obviates the need for separation steps or the labeling of antibodies in the performance of an assay. The tracer is a short antigen-mimicking peptide labeled with both a fluorescent energy transfer donor and fluorescent energy transfer acceptor molecules. When free in solution, the tracer exhibits very low fluorescence due to intramolecular dye dimerization. After binding to an antibody of the native antigen, fluorescence is significantly enhanced as a result of the dissociation of intramolecular dimers brought about by conformational changes in the tracer peptide.
The invention thus includes an immunoassay procedure for detecting and quantifying unknown analyte antibody or analyte antigen in bulk solution, a reagent for use in such an immunoassay procedure, and a process for making such a reagent. The reagent which is used in the present procedure is a peptide epitope that is recognized by the antibody in bulk solution, the peptide epitope conjugated to a pair of fluorescent energy transfer dyes capable of moving between a stacked configuration to exhibit fluorescence quenching and a spaced (unstacked) configuration to exhibit fluorescence.
A procedure for using the previously described reagent in a homogeneous antibody assay includes: placing in bulk solution a conjugate of a peptide epitope for the unknown amount of analyte antibody, and a pair of fluorescent dyes. This reagent is capable of moving between a stacked configuration to exhibit fluorescent quenching and a more spaced configuration to exhibit fluorescence. Also placed into the bulk solution is the unknown antibody which will bind with the peptide epitope which is conjugated to the pair of dyes. When this binding event occurs, the configuration of the pair of dyes will be altered between an initial stacked configuration (when in solution) to an unstacked configuration, when the epitope is bonded to the antibody, with a concomitant increase in fluorescent energy in response to the binding.
It is also within the contemplation of the invention to design a homogenous antigen assay or a homogenous DNA (or RNA) probe assay.
The tracers have uses including homogenous detection of macromolecules (e.g. antibodies, antigens, DNA, and RNA) of clinical interest and rapid screening of recombinant antibodies.